title: Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection.
abstract: The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS(Biotin)) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1J/cm(2) yielded an average conjugation efficiency of ≈ 1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBS(Biotin) method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH3(+) surface conjugation, NHS-Biotin, and direct physical adsorption. Taken together, the UV-NBS(Biotin) method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Nur
authors.initials: N
authors.lastname: Mustafaoglu
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D019921:Biotinylation; D007074:Immunoglobulin G / Q000737:chemistry*; D057230:Limit of Detection; D051379:Mice; D009711:Nucleotides / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000032:analysis*; D014466:Ultraviolet Rays
chemical_list: D055910:Antibodies, Immobilized; D007074:Immunoglobulin G; D009711:Nucleotides; D017430:Prostate-Specific Antigen
keywords: Biosensor; ELISA; Immunoglobulin; Oriented immobilization; Site-specific crosslinking